Historically, research on the actions of ethanol at the GABAergic synapse has focused on postsynaptic mechanisms. Recent data has challenged this view by demonstrating that ethanol also increases both spontaneous and evoked GABA release in many brain regions. However, little is yet known about the mechanism through which ethanol acts to enhance GABA release. Neurotransmitter release can be altered by changes in intracellular calcium levels, and intracellular calcium levels are increased by ethanol. Therefore, it seems plausible that calcium dependent mechanisms could play a role in ethanol-enhanced GABA release. Activation of inositol-1,4,5-trisphosphate receptors (IPSRs) and ryanodine receptors (RyRs) can influence neurotransmitter release by releasing calcium from internal calcium stores. Preliminary Data show that an IP3R inhibitor interferes with the ethanol-induced increase in miniature Inhibitory Postsynaptic Current (mlPSC) frequency in cerebellar Purkinje neurons (cPNs), suggesting that ethanol-enhanced GABA release depends on calcium release from internal calcium stores. I will explore this idea by confirming that IP3R inhibition reduces ethanol-enhanced GABA release and by determining whether RyRs are involved in ethanol-enhanced GABA release (Aim 1). Neuromodulators can alter ethanol-enhanced GABA release; one example being a cannabinoid (CB) receptor agonist, which prevents the ethanol-induced increase in mlPSC frequency in cPNs. The CB receptors are G protein-coupled receptors that act through G-alpha-i-linked second messengers; therefore, I will determine if ethanol is also acting through second messengers to alter the IPSRs and RyRs. There are protein kinase A (PKA) phosphorylation sites on both the IP3R and RyR, and ethanol and CBs both regulate PKA. Therefore, ethanol might influence IPSRs and/or RyRs through activation of PKA (Aim 2). Ethanol also alters phospholipase (PLC) activity. Because PLC generates IPS and leads to activation of protein kinase C (PKC), the mechanism of ethanol-enhanced GABA release could involve activation of the IP3R through production of IPS and/or phosphorylation of the IP3R by PKC (Aim 3). To thoroughly explore these Aims in cPNs, I will analyze mlPSC frequency, which is the most direct way to assess changes in neurotransmitter release. I will also use paired-pulse ratio (PPR), which can detect effects that occur more distal to the neurotransmitter release site. Overall, I will explore mechanisms that could account for the effect of ethanol on GABA release in the brain, a means by which ethanol can affect behavior. An answer to this important issue could assist the scientific community in understanding how this acute consequence of ethanol contributes to alcoholism. [unreadable] [unreadable] [unreadable]